Pregnancy test



Feb- 26, 1952 G. c. RICHARDSON x-:TAL 2,587,221

PREGNANCY TEST Filed MalCh 16, 1949 u........fnlgunlnn.l.HHN

, fn 067725725: Garwooa/ Cfz'cfzafdson Patented Feb. 26, 1952 'PREGNANCY TEST Garwood C. Richardson, Chicago, and Gustav W. Rapp, Glen Ellyn, Ill., assignors to Chicago Endocrine Research Company, Chicago, Ill., a corporation of Illinois Application March 16, 1949, Serial No. 81,828

4 Claims. (Cl. 23-230) The present invention relates to improved tests for determining pregnancy and includes improved apparatus for carrying out certain preferred pregnancy tests.

The art has long desired a simple, relatively inexpensive, rapid and accurate means for determining pregnancy in mammalia and particularly in women. The biological tests employed heretofore such as the rat, rabbit and frog, as well as the prostigmin skin tests, have proven complex and due to the complicated nature of the tests involved, to be relatively expensive and time consuming. Also, some of the tests available heretofore have been found to be lacking in desired accuracy, particularly during the early days of pregnancy.

The principal object ofthe present invention is to provide a simple, inexpensive, concise and accurate test for determining pregnancy, including early pregnancy in mammalia.

Another object of the present invention is to provide a rapid test for determining pregnancy which is capable of providing an accurate answer, confirming or ruling .out pregnancy, during the patients visit to the doctors oice, clinical laboratory or hospital.

A further object of the present invention is to provide a simple and inexpensive apparatus for carrying out certain preferred pregnancy tests.

Other objects of the present invention will be apparent as the vdetailed description proceeds hereinafter.

The improved tests employed in the present invention are based on the identification of estrone, a hormone associated with pregnancy, inthe urine from pregnant animals, e. g. cows, horses and dogs as well as women. There is a possibility, particularly during the menstrual cycle, that female urine may contain progesterone, another fied so as not to interfere with proper identication of estrone. We have discovered that this may be readily accomplished by the procedures described below.

Separation of progesterone The separation of progesterone and other interfering steroid substances from estrone in urine is accomplished by adding alkali to urineand extracting the resulting alkalinized urine with a waterimmiscible organic solvent. In this proc- It, therefore, follows, for an a ess the estrone to be identiiied remains in the aqueous layer or solution, and the progesterone, along with any other interfering steroid substances present in the urine, are taken up in the extract where they remain in the water irnmiscible layer-or organic solvent. In our pre'- ferred testing method, alkali metal hydroxide such as sodium or potassium hydroxide is used as the alkali and an organic solvent which is heavier or lighter than water such as chloroform, carbon tetrachloride, carbon disulphide, benzene, heptane or ether, is used as the water immiscible solvent. While the aqueous layer containing the estrone may be separated from the organic solvent layer by any suitable means as, for example, by decantation, by the use of` a separatory fun'- nel, etc., we prefer to use the simple and inexpensive extraction apparatus shown in the accompanying drawing.

The extraction apparatus comprises primarily an outer chamber or relatively large tube I0 and an inner tube or relatively small tube I3. The outer chamber is provided with a screw cap il and a mark identified at i2 as means for determining volumes of reagents to be used. The inner tube is provided with a hole or inlet opening I4, and the inner tube is of such size that it may be readily inserted into the outer chamber. The preferred method of using the extraction apparatus is described in detail in Example I.

Modification of progesterone The chemical modification vof progesterone, along with any other masking or interfering key tonic substances (pyruvic acid, acetone, etc.,) present in urine, is accomplished by adding alkalito urine and reactng the resulting alkalinized urine with` a water soluble hypohalide,- e. g. alkali metal hypoioddes, hypochlordes, etc. In our preferred testing method alkali metal hydroxide such as sodium or potassium hydroxiide is used as thealkali and the alkalinized urine is reacted with anl aqueous iodine/potasl sium iodide solution or aqueous chlorine/potassium chloride solution. In this process it is not necessary to separate progesterone from estrone as the progesterone and other ketonic bodies in their modified form do not interfere with proper identification of estrone. The estrone remains unchanged in the hypohalide reaction mixtureand may readily be identified as described'below.

The tests employed in the present invention are based upon the discoveries (1) that estrone reacts with 2,4-dinitrophenylhydrazine in an acid medium which, when made alkaline, gives a" stable brown color and (2) that estrone reacts v.with meta-dinitrobenzene in alkaline solution to give a stable red or reddish violet color. Pref ferred methods of carrying out the tests are described below in the -illustrative examples.

EXANIPLE I Withfa calibrateddropper, place12m1. ofrurine to be tested into outer chamber l!) of the ex traction apparatus and then add 2 drops of 0.5 normal sodium hydroxide. To the alkali-urine mixture next add about 2 ml. of U. S. P. `chloroform, i. e. sufficient to bring the .total volume up to mark l2 on chamber IB. Place screw'cap Il on chamber Il] and shake vigorously for -at least about 30 seconds and then allow to stand quietly for about one minute or until the two layers (chloroform and aqueous layer) have separated sharply. Remove the screw cap and insert inner tube I3 slowly into the outer cham- .ber until the tube is completely inserted. Duringthisoperation the upper aqueous layer containing `any estrone present Yflows vthrough hole r inlet opening l!! into inner tube I3. The outerchamber and inner tube are next'separated and .the chloroform, with a small amount io'f .remaining `aqueous solution in the chamber, Vis discarded. 'I'he chamber is then r4rinsed well Withwater.

.Aboutone-'half of the aqueous solution in the inner rtube is .poured into `the washed outer chamber and to this solution is added 4 drops of ,0.5 fnormal sulfuric acid withJmixing. 'Iothis mixture is next added one ml. of 0.1 percent 2,4-dinitrophenylhydrazine .in ethanol, `and the resulting mixture allowed to stand for "about 1) to 1.5 minutes. "The reaction mixture is then mader alkaline by the addition of one ml. o1 0.5 normal sodium hydroxide. If thewtest is negative andestrone is notv present inithetest sample, the resulting brown color usually 'fades within two minutes. .If .the'brown. color remains forat least aboutve minutes (usually'to l'minutes orglonger) Athen estrone is `present andthe itest is .positivejfor pregnancy.

EXAMPLEEII 'The procedureis the same'as'that described rin E'xarr-1ple I up to :and including the 4pou-ring Aoi `aboutgone-hali of Y"the aqueous'solutionthe inner *tube into'the washed"outenchambeiz To the solution inthe outer chamber is added 5 drops of one per cent meta-nitrobenzene in ethanol, with mixing. About one ml. of 1'5 percent potassium hydroxideis` then added and theresulting'mixture allowed to stand for'about AI() minutes. If noreddish violet ycolor is observed, fthe testis negative, while if thesolution Ihasf'turnel red'or reddish violet the test is positive ifor'pregnancy.

The method employed in 'Example' -II may Ibe advantageously combined with the methodlemployed in Example I. When this is done, ya double check for the presence of estrone is obtained by testing one-'half of the aqueous solu- 'tion with 2,4-dinitrophenylhydrazinein accordance Vwith Example I, and by testing the other half yof the aqueous solution with metanitrobenzene in accordance with Example II.

EXAMPLE III With a calibrated dropper, place 2 ml. of urine to be tested into the outer chamber of the extraction apparatus or other VVsuitable container, andthen'add 2 drops of 0.5 normal'scdium'hydroxide with mixing. V'I'ov thevalkali-urine mixtureis added drops ofan aqueous 2 per cent iodine/4 per cent` potassium iodide solution with mixing, and the resulting Vreaction mixture is allowed to stand for approximatelyione min-nte. To this mixture is AJnext `added with mixing e mixture.

iinal reaction mixture.

drops of 0.5 normal sulfuric acid followed by 1 ml. of 0.1percent 2,4-dinitrophenylhydrazine in ethanol. The resulting reaction mixture is allowed to stand for about l0 to 15 minutes, and

Lthen one ml. of 0.5 normal sodium hydroxide is added., Ii the test is negative and estrone is not present in the test sample, the brown color formed in the .solution usually fades Within two minutes. If the brown color remains for at least about iiveminutes (usually 5 to 10 minutes or longer), then estrone is present and the test is positive for pregnancy.

EXAMPLE IV The lprocedure is the same as that described `in Example, III up'toand including'thel addition of the iodine/potassium iodide solution. To-'this mixture is added 5 drops of 1 per centmetanitrobenzene in ethanol, with mixing. About one ml. of l5'per cent potassium hydroxide is then `added `with mixing and thev resulting' reactionmixture allowed to stand vfor about'tenmin'- utes. If no reddish violet color is observed, -the test is negativefwhile' if the solution has turned reddish lviolet in color, the test is positive :for

pregnancy.

The `procedure in Example'IV may also'b'ecombined with the procedure-'describedin Example III to 'give adouble check `for positive'or negative results. n'Examples II and IV the initial'addition of alkali makes the urine solution slightly alkaline, while the nal 'addition of alkali makes kother acids may also be used to providethe desired acidic reaction mixtures, although -it `'is preferred to employ stable mineral acids fsu-ch as sulfuric acid, hydrochloric acid 'andthe "like which are relatively cheap yand*readilyavailable in the open'market. The use ofthe halogen/alkali metal halide mixturefin alkaline solution -is a particularly convenient means for introducing the water soluble hypohalide into the reaction In Examples III and IV above employing iodine and lpotassium io'dide in the Vurine alkalinized with -sodium hydroxide, fthe hypo- 'halide is present as sodium'hypoio'dide.

Any water immiscible organic 'solvent may'be used in the processes described 'in Examples '-I and 'IL although the Ause of Vsolvents with :about the same speci'c gravity as the aqueous urine is not'preferred as they tend to form emulsions with the urine which donotfseparate 'rapidly'into twolayers. Whenasolvent such as etherfor heptane (which are lighter than the 'aqueous urine) is used inplacef aheavierlsclvent such 'as chloroform used in'EXamplesI `and L-the'extraction apparatus shown in the drawingsmay still be used, but in this case Lthe upper organic solvent layerrcontaining the interferingsteroid material ilows through-hole `lfd intoth'e 'inner `tube i3 and is discarded, andthe 'tests y'a1-econducted with the aqueous solution 'remaining in the outer chamber.

4The present invention'provides simpler-and expensive tests for determining pregnancy of the type long desired. Instead of hours or even days required for the old biological tests available heretofore, the novel chemical tests of the present invention may be easily completed in twenty minutes, and several tests may be readily run at the same time. In practice the methods of the present invention have also provenin well over a thousand tests, to be 100 per cent accurate in confirming or ruling out pregnancy.

The apparatus In the drawing there is illustrated one form of very simple, inexpensive, and easy to use apparatus which is especially adapted to the carrying out of tests in accordance with the foregoing Examples I and II, especially the step of separating immiscible liquids.

In the drawing:

Fig. 1 is a side elevation except that upper and ceptacle I0 is of generally cylindrical form, and

the contracted mouth end portion I6 is likewise of cylindrical form. The cap I I may be provided with a gasket I 8 which is adapted to form a leakproof closure for the mouth end of the receptacle when the cap I I is applied to the receptacle. The

able manner.

The inner receptacle I3 is also preferably formed of transparent glass and in the form of an ordinary test tube. The outside diameter of the receptacle I3 is somewhat less than the internal diameter of the body portion of the outer receptacle I0 so that said inner receptacle I3 will t freely within the receptacle I0. The outer diouter receptacle. It will, of course, be understood that the telescopic receptacles are so proportioned in size that the space remaining within the two receptacles when telescoped, as shown in Fig. 3, will be sufficient to contain the lower layer of immiscible liquids without elevating the same high enough to flow through the opening I4 in the inner tube. Hence, when the iiow of liquid into the inner tube has ceased, the inner tube may be withdrawn from the outer tube and used in the manner of an ordinary test tube for further testing operations.

The flange I9 of the inner receptacle is of such size that the cap II may be applied to the assembled structure so that the cap may be employed to clamp the inner receptacle in iixed position in the outer receptacle.` In this manner, the testing apparatus is nested for packaging and transport as a completely packaged unit, the cap also serving to prevent the entrance of foreign material into the receptacles. If desired, a suitable gasket may be placed around the inner receptacle immediately under its mouth end flange, to cushion the contact between said ange and the end of the outer receptacle.

The described apparatus, being of Simple constructional details may be produced at very low cost. Furthermore, the employment of the described apparatus requires no high degree of care and is, therefore, well adapted to use in the carrying out of tests to be made in a matter of minutes rather than hours or longer periods. Also, the apparatus is such that it may properly be used with a minimum of explanation or inameter of the receptacle I3 is, however, a close butl t easy sliding fit in the reduced diameter mouth portion I6 of the outer receptacle so that said mouth portion will effectively center the tube I3 in the outer receptacle when the two receptacles made of substantial length as shown. The upper or mouth end of the inner receptacle I3 is provided with an outwardly extending flange I9. which may seat on the mouth end of the outer receptacle as shown at Fig. 3 to limit the extent to which the said receptacle I3 may be inserted.

in the outer receptacle.

The inner receptacle I3 is provided with the opening I4 in its side wall at a predetermined distance from the closed bottom end 20 of the (receptacle. When the inner receptacle I3 is telescoped into the outer receptacle as shownarate such uppermost layer of liquid from the.

heavier lower layer which will remain in the struction and without practice or experience.

Changes may be made in the apparatus while retaining the principles of the described strucwall of the receptacle II) is provided with theg'f' gauge mark I2 by etching or in any other suit# ture, and, while the described apparatus is preferred because of its simplicty, it is not essential to the practice of the testing methods hereinbefore described. `Other liquid separation apparatus and procedures may be eiectively employed.

We claim:

l. In a method for determining pregnancy the steps which comprise, alkalinizing femaleurine, extracting the alkaline urine with a water immiscible organic solvent, separating the aqueous phase of the extracted urine from the organic solvent extract, acidifying the extracted urine,

, adding 2,4-dinitrophenylhydrazine to the acidined urine, and alkalinizing the resulting reaction are assembled as shown in Fig. 3. To that end, 'mlxture' the mouth portion I6 of the outer receptacle isA Gig? 2. In a method for determining pregnancy the steps which comprise, adding sodium hydroxide to female urine, extracting the alkaline urine with chloroform, separating the aqueous phase of the extracted urine from the chloroform extract, acidifying the extracted urine with sulfuric acid, adding 2,4-dinitrophenylhydrazine to the acidiiied urine, and adding sodium hydroxide to alkalinize the resulting reaction mixture.

3. A test for determining pregnancy based on the identification of estrone in urine which comprises, alkalinizing female urine by addition of alkali metal hydroxide, extracting the alkaline urine with a water immiscible organic solvent, separating the resulting aqueous urine layer from the organic solvent extract, acidifying the urine, adding 2,4-dinitrophenylhydrazine to the acidiied urine, alkalinizing the reaction mixture by addition of alkali metal hydroxide, and then observing the resulting brown color formation in the alkaline reaction mixture, said test being positive for the presence of estrone when the 'b1-own,` ,color remains in the reaction fmixture f or :at ieastzabout ve minutes.

4.A-;test;for determining pregnancy based on the identification' of estrone zin urine which comprises,:a1ka1inizing female urine by `addition of sodium hydroxide, extracting ,the alkaline-urine with chloroformfseparating the-resulting aqueous urine layer 'from the chloroform extract, ,aodfyngathe urine Witheulfuric acd,..adding,2,4

dntrophenylhydrazine ,to vthe i acidied urine, 10

alkalinizing the reaction mixture by addition of sodium hydroxide, and then observing there- 4sulting -brown color .formation in Athe valkaline reaction mixture, `said, test being postivefor ,the

presence .fof-estrone when the brown Ycolor .,re- ,1,5 "2.571072 REFERENCES CITED '-.The following rre'ferences @are of record inithe 'le ofathis patent:

Number Name Date 1,722,160 Smith July 23, 1929 1,968,131 Edwards July 31, 1934 '2,471,861 :Cahn "May"'31, "1949 Maurer Ju'1y2'6, 1949 

1. IN A METHOD FOR DETERMINING PREGNANCY THE STEPS WHICH COMPRISE, ALKALINIZING FEMALE URINE, EXTRACTING THE ALKALINE URINE WITH A WATER IMMISCIBLE ORGANIC SOLVENT, SEPARATING THE AQUEOUS PHASE OF THE EXTRACTED URINE FROM THE ORGANIC SOLVENT EXTRACT, ACIDIFYING THE EXTRACTED URINE, ADDING 2,4-DINITROPHENYLHYDRAZINE TO THE ACIDIFIED URINE, AND ALKALINIZING THE RESULTING REACTION MIXTURE. 